Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum
Abstract Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2024-02-01
|
| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-024-05906-2 |
| _version_ | 1797273550173765632 |
|---|---|
| author | Yunyi Gao Therese Solberg Rui Wang Yueer Yu Khaled A. S. Al-Rasheid Feng Gao |
| author_facet | Yunyi Gao Therese Solberg Rui Wang Yueer Yu Khaled A. S. Al-Rasheid Feng Gao |
| author_sort | Yunyi Gao |
| collection | DOAJ |
| description | Abstract Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future. |
| first_indexed | 2024-03-07T14:45:59Z |
| format | Article |
| id | doaj.art-b5b7275faf7d49268125810730a6d921 |
| institution | Directory Open Access Journal |
| issn | 2399-3642 |
| language | English |
| last_indexed | 2024-03-07T14:45:59Z |
| publishDate | 2024-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj.art-b5b7275faf7d49268125810730a6d9212024-03-05T20:00:04ZengNature PortfolioCommunications Biology2399-36422024-02-017111110.1038/s42003-024-05906-2Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatumYunyi Gao0Therese Solberg1Rui Wang2Yueer Yu3Khaled A. S. Al-Rasheid4Feng Gao5Key Laboratory of Evolution & Marine Biodiversity (Ministry of Education), and Institute of Evolution & Marine Biodiversity, Ocean University of ChinaDepartment of Molecular Biology, Keio University School of MedicineKey Laboratory of Evolution & Marine Biodiversity (Ministry of Education), and Institute of Evolution & Marine Biodiversity, Ocean University of ChinaKey Laboratory of Evolution & Marine Biodiversity (Ministry of Education), and Institute of Evolution & Marine Biodiversity, Ocean University of ChinaZoology Department, College of Science, King Saud UniversityKey Laboratory of Evolution & Marine Biodiversity (Ministry of Education), and Institute of Evolution & Marine Biodiversity, Ocean University of ChinaAbstract Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.https://doi.org/10.1038/s42003-024-05906-2 |
| spellingShingle | Yunyi Gao Therese Solberg Rui Wang Yueer Yu Khaled A. S. Al-Rasheid Feng Gao Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum Communications Biology |
| title | Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum |
| title_full | Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum |
| title_fullStr | Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum |
| title_full_unstemmed | Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum |
| title_short | Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum |
| title_sort | application of rna interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan paramecium caudatum |
| url | https://doi.org/10.1038/s42003-024-05906-2 |
| work_keys_str_mv | AT yunyigao applicationofrnainterferenceandproteinlocalizationtoinvestigatehousekeepinganddevelopmentallyregulatedgenesintheemergingmodelprotozoanparameciumcaudatum AT theresesolberg applicationofrnainterferenceandproteinlocalizationtoinvestigatehousekeepinganddevelopmentallyregulatedgenesintheemergingmodelprotozoanparameciumcaudatum AT ruiwang applicationofrnainterferenceandproteinlocalizationtoinvestigatehousekeepinganddevelopmentallyregulatedgenesintheemergingmodelprotozoanparameciumcaudatum AT yueeryu applicationofrnainterferenceandproteinlocalizationtoinvestigatehousekeepinganddevelopmentallyregulatedgenesintheemergingmodelprotozoanparameciumcaudatum AT khaledasalrasheid applicationofrnainterferenceandproteinlocalizationtoinvestigatehousekeepinganddevelopmentallyregulatedgenesintheemergingmodelprotozoanparameciumcaudatum AT fenggao applicationofrnainterferenceandproteinlocalizationtoinvestigatehousekeepinganddevelopmentallyregulatedgenesintheemergingmodelprotozoanparameciumcaudatum |